Heparine derivatives and method of making same



ponding alkali metal salt of benzyl heparinate.

United States Patent Ic 3,210,250

Patented Oct. 5, 1965 3 210 250 The high molecular weight long chain quaternary am- HEPARINE DEmfATlvErs AND METHOD monium compounds used to form the corresponding salt OF MAKING SAQWE of heparine are such as, for example, benzyldimethyl Robert Buconrt, Clichy-sous-Bois, Seine et Oise, France, 2 (P 1, y y -p y)- y]- assignor to Roussel-UCLAF, Paris, France, a corpora- 5 ethyl ammonium chloride, which is also known under the tion of France trademark Hyamine 1622 trimethyl (methyldodecyl- N0 D n Filed 5 1964, Sen ,010 benzyl)-ammonium chloride dilauryldimethylammonium Claims Pl'lOllty, appl cation France, Jan. 18, 1963, chloride and other suitable compounds.

9 (g A preferred mode of the process of the invention 10 comprises reacting benzyl bromide with the Hyamine The invention relates to the novel compound, benzyl 1622 Salt of heparine in tetrahy to f rm the heparinate and its alkali metal salts and to a novel proc- Hyamine 1622 Salt f n y h parinate a rea ting the ess for its preparation. The invention further relates to latter with an aqueous Solution of Sedhlm acetate to form novel anticoagulating compositions having an elevated the Sodium Salt of benzyl hePaTihate Whieh can be P p antilipemic and clarifying activity and to a novel method teted from Solution y the addition of a lower alkanol 0f retarding the coagulation of blood. such as methanol. Other alkali metal salts such as the It is an object of the invention to provide the novel Potassium Salt can be formed y using the Potassium compound, benzyl heparinate and it alk li met l lt salt of lower alkanoic acids in place of sodium acetate.

It is another object of the invention to provide a novel AS has already been indicated above, benzyl parinate process fo the preparation f benzyl h l t d and its alkali metal salts possess particularly the advanits alkali metal salts, tage over heparine of exercising a prolonged action which It is a further object of the invention to provide novel avoids repeated intravenous injections Continuous anticoagulating compositions which have an elevated Venous Pertusiens required With Very elevated doses of antilipemic and clarifying a ti heparine. It possesses, in addition, the advantage over It is an additional obje t of th i ti t id anticoagulants derived from dicoumarine of acting with a novel method of retarding coagulation of blood. p y and during an easily determinable y, Where- These and other objects and .advantages of the invention as the derivatives f ll r Il Which inhibit the will become obvious from the following detailed descrip- Synthesis of thrembhle in the liver (antagonists of Vitetion. min K) have a delaying action but whose duration cannot The novel compound of the invention is benzyl hepabe foreseen With eertitllde' y, for ahght We knew, rinate and its alkali metal lt whi h h th b bl the action of benzyl heparinate and its alkali metal salts structural formula is manifest on all the stages of coagulation. This ren- COOCH2C5H5 CH OSO R $OOOH2C6H5 C|H2OSO3R OSOaR OH OH OH 0 \i l/ 0 0H l qHsosR 3 OH NHSO R 11 wherein R is Selected from the group consisting of hyders their action more sure and less dangerous for the drogen and an alkali metal. 40 organism.

Some derivatives of heparine which possess prolonged The hove1 ahtieeagulattng Compositions Of the v nanticoagulating activity are known. Benzyl heparinate tieh Which Possess an elevated ahtihpemie and clarifying and its alkali metal salts, however, possess a prolonged activity are Comprised of a Compound Selected from the anticoagulating activity which is marked at relatively low 8 P Consisting of benzyl heparinete its alkali metal doses and also possess an elevated antilipemic and clarify- Salts and a major amount of a Pharmaceutical Carriering activity. The said compositions may be in the form of aqueous The process for the preparation of an alkali metal salt inieetable Solutions and Suppositories P p in the of benzyl heparinate comprises reacting a benzyl halollsllal mannergenide with a high molecular weight long chain quater- The novel method of the invention of retarding the nary ammonium salt of heparine in an inert organic 501- coagulation of blood comprises administering an effecvent to form the corresponding quaternary ammonium tiVe amount of a compound Selected from the group e011- salt of benzyl heparinate which is reacted with an alkali Sistihg Q benzyl hepafhlate and its alkali al tsnetal salt of a lower alkanoic acid to form the corres- The eelhpeuhds y be administered r n n ously by intramuscular, subcutaneous or intravenous injections The process for the preparation of benzyl heparinate or rectany' h usual dally dosage is 9 9 comprises reacting a benzyl halogenide with a high molecper day dependlpg upon the method of admliustrauon' ular weight long chain quaternary ammonium salt of In the followlpg example there are .descnlmd Several heparine in an inert Organic solvent to form the Corre preferred embodiments to illustrate the lnvention. However, it should be understood that the invention is not spondlng quaternary ammonium salt of benzyl heparinate, 0 intended to be limited to the specific embodiments which is reacted with an alkali metal salt of a lower alkanoic acid to form the corresponding alkali metal Example I- Prelmmti0 the Sodium Saltof salt of benzyl heparinate, the aqueous solution of which benzyl hepa'mate is treated by an acid ion exchange resin to form benzyl 11 grams of the neutral heparine salt of Hyamine 1622 heparinate. (obtained according to the United States Patent No.

2,989,438) and 110 cc. of tetrahydrofuran were introduced into a three-necked balloon flask and the mixture was agitated until total dissolution occurred. Thereafter, 55 cc. of benzyl bromide were added and the mixture was agitated and the tetrahydrofuran was distilled therefrom. Precipitation was brought about by the addition of 400 cc. of methanol. The benzyl heparinate was separated by filtration, vacuum filtered and washed with methanol. The precipitate was taken up with 80 cc. of an aqueous solution containing 20% sodium acetate and 300 cc. of methanol were added thereto. The benzyl heparinate precipitated therefrom in the form of its sodium salt. The precipitate was vacuum filtered and washed with methanol. The precipitate was then redissolved in water and filtered through carbon black and infusorial earth. The filtrate was poured into a mixture of 450 cc. of methanol and 50 cc. of a methanolic solution of sodium acetate. The precipitate was separated, vacuum filtered, washed with methanol and dried to obtain 2.993 gm. of benzyl heparinate in the form of its sodium salt. The product occurred in the form of an amorphous powder of white color, and was soluble in water, very slightly soluble in alcohol and insoluble in ether, acetone, benzene and chloroform. Its specific rotation [a] =+33.3i0.5 (c.=l% in water).

Analysis.RaW formula, [C7 H3gO73N4S11Na1 I molecular weight=(2832.23) Calculated: C, 32.22%; H, 3.16%; N, 1.97%; ash, 27.5. Found: C, 32.332.l%, H, 3.33.3%; N, 1.8l.9%; ash, 27.5-27.5.

U.V. spectra:

These results correspond approximately to benzyl groups about 2.2 10 molecules per gram. The product had an anticoagulant activity in vitro of 22 U/rng. and an antilipemic activity in vitro of 128 U/mg.

This compound is not described in the literature.

Pharmacological study of benzyl heparinate and its alkali metal salts (1) Immediate anticoagulant activity in vitr0.-A plasma reactant deprived of calcium and of prothrombine was prepared. By adding thrombine to this plasma, the fibrinogen which it contained was transformed into fibrin which produced simultaneously a coagulation and an opacification. The previous addition of an anticoagulant such as hepan'ne provokes a retardation of the coagulation and of the opacification. If the time necessary in order to obtain a given value of opacity is measured, it can be observed that there exists a relation between this time and the coagulating activity of the preparation. The values obtained are reported as those found with a solution titrated with heparine as the control. These values are expressed in antithrombic units (U.A.T.).

RESULTS U.A.T./mg. Heparine control (sodium salt) 130-140 The sodium salt of beuzyl heparinate 22 (2) Delayed anticoagulant activity in vivo by inimvenous methd.This action was studied in the rabbit by determining the time of coagulation at regular intervals (2, 4, 6, 8, hours after intravenous injection of the compound studied). The compound was admin- As can be seen from the examination of this table, the sodium salt of benzyl heparinate possesses a prolonged anticoagulant action at anticoagulant doses (expressed in units) much lower than those of heparine necessary to obtain an eitect of the same order.

(3) Clarifying activity in vitro.This activity was studied by utilizing the technique published by Plotka et al. (Arch. Int. Pharmacodyn, 1960, 126, After having verified on several samples of heparine that the variation of anticoagulant and antilipemic values were always parallel, a heparine testing anticoagulant units per mg. was taken as a reference standard to which the value of 150 clarifying or antilipemic units was arbitrarily assigned to determine the antilipemic activity of the sodium salt of benzyl heparinate in antilipemic units. To this effect the variations of turbidity caused by the action of increasing doses of standard heparine were measured in the presence of CaCl on the same volume of the artificial substrate of egg yellow. In certain limits of concentrations of heparine, the graphic representation was a straight line. The test was repeated on the sodium salt of beuzyl heparinate and with reference to the graph of the reference compound, the value in antilipemic units (A.L.) of the sample was found to be 128 U./mg.

(4) Determination of t0xicity.The sodium salt of benzyl heparinate placed in solution in physiological serum was administered to lots of ten mice of the Rockland strain weighing between 18 and 22 gm. The medicine was injected intravenously in a volume of 10 cc. per kg. of animal at doses of 100 mg./kg. and 200 mg./kg.

The animals were held under observation for a period of one week. At a dose of 200 mg./kg., four mice out of ten died. No mortality was noted at a dose of 100 mg./ kg. Therefore, the sodium salt of beuzyl heparinate in acute testing on mice at a dose of 100 mg./kg. admin istered intravenously is devoid of toxicity.

Various modifications of the compositions and process of the invention may be made without departing from the spirit or scope thereof and it is to be understood that the invention is to be limited only as defined in the appended claims.

I claim:

1. A compound selected from the group consisting of benzyl heparinate and its alkali metal salts.

2. Benzylheparinate.

3. The sodium salt of beuzyl heparinate.

4. A process for the preparation of an alkali metal salt of benzyl heparinate which comprises reacting a beuzyl halogenide with a high molecular weight quaternary ammonium salt of heparine in an inert organic solvent to form the corresponding quaternary ammonium salt of benzyl heparinate which is reacted with an alkali metal salt of a lower alkanoic acid to form the corresponding alkali metal salt of benzyl heparinate.

5. The process of claim 4 wherein the beuzyl halogenide is benzyl bromide.

6. A process for the preparation of the sodium salt of benzyl heparinate which comprises reacting benzyl bromide with a high molecular weight quaternary ammonium salt of heparine in an inert solvent to form the corresponding quaternary ammonium salt of benzyl heparinate and reacting the latter with an aqueous solution of a sodium salt of a lower alkanoic acid to form the sodium salt of benzyl heparinate.

7. A process for the preparation of benzyl heparinate, which comprises reacting a benzyl halogenide with a high molecular Weight quaternary ammonium salt of heparine in an inert organic solvent to form the corresponding quaternary ammonium salt of benzyl heparinate, which is reacted with an alkali metal salt of a lower alkanoic acid to form the corresponding alkali metal salt of benzyl heparinate, the aqueous solution of which is treated by an acid ion exchange resin to form benzyl heparinate.

8. An anticoagulant composition comprised of a compound selected from the group consisting of benzyl heparinate and its alkali metal salts and a major amount of a pharmaceutical carrier.

9. The composition of claim 8 wherein the said compound is the sodium salt of benzyl heparinate.

No references cited.

JULIAN S. LEVITT, Primary Examiner. 

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF BENZYL HEPARINATE AND ITS ALKALI METAL SALTS. 